Obesity continues to increase as a public health emergency, with the origins of most adult obesity being in childhood. Effective clinical approaches are urgently needed to prevent or reverse childhood obesity. The gut-associated microbiome is an established central factor in energy harvest, hepatic function, insulin sensitivity, and adipose tissue homeostasis, making it a critical target for obesity intervention strategies.
This project represents a collaborative effort between Lurie Children’s Hospital and Abbott Nutrition to investigate interpersonal variation in energetics and short-chain fatty acid (SCFA) production of obesity-associated gut microbiota in response to slow and fast digestible carbohydrates. The work builds upon foundational research demonstrating that carbohydrate quality, rather than quantity alone, plays a crucial role in metabolic health.
The project was motivated by compelling evidence from the Frontiers in Nutrition paper by Wang et al. (2022) that demonstrated the therapeutic potential of slowly digestible carbohydrates (SDC) in metabolic syndrome and obesity management. This seminal work showed that SDC displays beneficial effects on reducing glucose excursions in healthy, insulin-resistant, and type 2 diabetic individuals, inducing a slow and prolonged glucose release that results in reduced postprandial glycemic responses and extended glycemic index values.
In type 2 diabetic patients, SDC-rich diets (60g/day) reduced glycemic variability parameters by 17-23%, with these parameters correlating with HbA1c, suggesting potential for long-term glycemic improvement. The Frontiers paper also demonstrated that foods with the highest SDC content (23.9-27.5 g/100g) induce the lowest glycemic responses with the lowest incremental AUC of glucose and insulin concentration.
In comparison to fast digestible carbohydrate (FDC) sources, the Abbott group has shown in murine models of obesity that nutrition with slow digestible carbohydrates (SDC) reverses obesity-associated phenotypes, including elevated body mass, insulin resistance, and systemic inflammation. However, the interpersonal differences in SDC responses by the childhood-associated human microbiota may not be fully predicted in murine obesity models.
Lurie investigators and colleagues have shown interpersonal variation in the production of short-chain fatty acids (SCFA) by the human gut microbiome from adolescents with obesity in response to ex vivo prebiotic exposure, suggesting that complex carbohydrate utilization by the microbiota varies between individuals and thus may affect who responds to SDC and other nutritional approaches to obesity.
Understanding the variation in the compositional and metabolic responses of the childhood-associated microbiota may inform future obesity-treatment trials and precision approaches to obesity therapy.
The main objectives of this project are to:
Understanding the interpersonal differences in SDC utilization by the childhood-associated human microbiota may inform future obesity-treatment trials through the identification of likely responders and, subsequently, precision approaches to obesity therapy. This precision nutrition approach could revolutionize childhood obesity treatment by enabling personalized dietary interventions based on individual microbiome composition and metabolic capacity.
This document presents the analysis of short-chain fatty acids (SCFAs) in the Abbott carbohydrate obesity project. The analysis examines how SCFA analytes change between experimental groups, carbohydrate types, and time points, with particular focus on identifying interpersonal variation in metabolic responses to different carbohydrate sources.
The fecal metabolome was analyzed using targeted metabolomics approaches. The DFI Host-Microbe Metabolomics Facility (DFI-HMMF) analyzed fecal material using validated methods and analysis pipelines. All compounds were validated through retention time and fragmentation comparison to standards and available databases.
Short chain fatty acids were analyzed using Gas chromatography-mass spectrometry (GC-MS) following derivatization with pentafluorobenzyl bromide (PFBBr). SCFAs (acetate, butyrate, propionate) were quantitatively analyzed following PFB derivatization and detection by negative collision induced gas chromatography-mass spectrometry ((-)-CI-GC-MS, Agilent 8890). Additional compounds including 5-aminovalerate and succinate were also quantified.
The following section outlines the specific protocol used for SCFA derivatization and GC-MS analysis.
Short chain fatty acids were derivatized as described by Haak et al. with modifications. The metabolite extract (100 µL) was added to 100 µL of 100 mM borate buffer (pH 10), 400 µL of 100 mM pentafluorobenzyl bromide in Acetonitrile, and 400 µL of n-hexane in a capped mass spec autosampler vial. Samples were heated to 65°C for 1 hour while shaking at 1300 rpm. After cooling, samples were centrifuged at 4°C, 2000 x g for 5 min, allowing phase separation. The hexanes phase was transferred and analyzed.
Samples were analyzed using a GC-MS (Agilent 7890A GC system, Agilent 5975C MS detector) operating in negative chemical ionization mode, using a HP-5MSUI column (30 m x 0.25 mm, 0.25 µm), methane as the reagent gas and 1 µL split injection (1:10 split ratio). A 10-point calibration curve was prepared with acetate (100 mM), propionate (25 mM), butyrate (12.5 mM), and succinate (50 mM), with 9 subsequent 2x serial dilutions.
This section describes the procedure for extracting metabolites from the fecal samples prior to analysis.
Extraction solvent (80% methanol spiked with internal standards and stored at -80°C) was added at a ratio of 100 mg of material/mL of extraction solvent. Samples were homogenized at 4°C on a Bead Mill 24 Homogenizer, set at 1.6 m/s with 6 thirty-second cycles, 5 seconds off per cycle. Samples were then centrifuged at -10°C, 20,000 x g for 15 min and the supernatant was used for analysis.
## Dataset dimensions: 440 16
## Sample groups: Case Control
## Carbohydrate types: Rapid Digestible Slow Digestible No Carbohydrate
## Time points: 0 48
## Total observations after averaging technical replicates: 950
| Group | Analyte | n | Mean | Median | SD | SEM | Q25 | Q75 |
|---|---|---|---|---|---|---|---|---|
| Case | 5aminovalerate | 94 | 0.476 | 0.158 | 0.613 | 0.063 | 0.050 | 0.776 |
| Control | 5aminovalerate | 96 | 0.549 | 0.415 | 0.539 | 0.055 | 0.050 | 1.070 |
| Case | acetate | 94 | 17.054 | 1.842 | 16.936 | 1.747 | 1.177 | 30.771 |
| Control | acetate | 96 | 18.722 | 21.415 | 17.680 | 1.804 | 1.182 | 33.516 |
| Case | butyrate | 94 | 3.513 | 0.555 | 3.870 | 0.399 | 0.259 | 6.370 |
| Control | butyrate | 96 | 4.205 | 3.350 | 4.408 | 0.450 | 0.270 | 7.136 |
| Case | propionate | 94 | 2.651 | 0.497 | 2.983 | 0.308 | 0.170 | 4.615 |
| Control | propionate | 96 | 2.736 | 1.982 | 2.862 | 0.292 | 0.159 | 5.366 |
| Case | succinate | 94 | 0.746 | 0.152 | 1.321 | 0.136 | 0.000 | 0.726 |
| Control | succinate | 96 | 1.254 | 0.270 | 1.885 | 0.192 | 0.000 | 1.646 |
| Carbohydrate Type | Analyte | n | Mean | Median | SD | SEM | Q25 | Q75 |
|---|---|---|---|---|---|---|---|---|
| No Carbohydrate | 5aminovalerate | 62 | 0.549 | 0.140 | 0.582 | 0.074 | 0.050 | 1.045 |
| Rapid Digestible | 5aminovalerate | 64 | 0.485 | 0.288 | 0.566 | 0.071 | 0.050 | 0.748 |
| Slow Digestible | 5aminovalerate | 64 | 0.506 | 0.338 | 0.590 | 0.074 | 0.050 | 0.826 |
| No Carbohydrate | acetate | 62 | 15.630 | 10.445 | 15.034 | 1.909 | 1.200 | 29.942 |
| Rapid Digestible | acetate | 64 | 18.221 | 22.682 | 17.598 | 2.200 | 1.169 | 32.874 |
| Slow Digestible | acetate | 64 | 19.770 | 23.908 | 18.964 | 2.370 | 1.183 | 37.334 |
| No Carbohydrate | butyrate | 62 | 3.104 | 1.750 | 3.013 | 0.383 | 0.280 | 6.070 |
| Rapid Digestible | butyrate | 64 | 3.975 | 2.243 | 4.599 | 0.575 | 0.262 | 6.670 |
| Slow Digestible | butyrate | 64 | 4.485 | 3.790 | 4.567 | 0.571 | 0.270 | 7.875 |
| No Carbohydrate | propionate | 62 | 3.097 | 1.610 | 3.188 | 0.405 | 0.172 | 5.958 |
| Rapid Digestible | propionate | 64 | 2.238 | 1.825 | 2.404 | 0.301 | 0.160 | 3.809 |
| Slow Digestible | propionate | 64 | 2.759 | 1.650 | 3.082 | 0.385 | 0.168 | 5.220 |
| No Carbohydrate | succinate | 62 | 0.638 | 0.235 | 1.046 | 0.133 | 0.000 | 0.690 |
| Rapid Digestible | succinate | 64 | 1.075 | 0.225 | 1.718 | 0.215 | 0.096 | 1.549 |
| Slow Digestible | succinate | 64 | 1.283 | 0.238 | 1.976 | 0.247 | 0.000 | 1.980 |
| Time (Hours) | Analyte | n | Mean | Median | SD | SEM | Q25 | Q75 |
|---|---|---|---|---|---|---|---|---|
| 0 | 5aminovalerate | 96 | 0.088 | 0.050 | 0.152 | 0.015 | 0.050 | 0.070 |
| 48 | 5aminovalerate | 94 | 0.947 | 0.840 | 0.525 | 0.054 | 0.595 | 1.191 |
| 0 | acetate | 96 | 2.484 | 1.180 | 7.620 | 0.778 | 0.990 | 1.335 |
| 48 | acetate | 94 | 33.638 | 32.780 | 7.253 | 0.748 | 29.030 | 38.329 |
| 0 | butyrate | 96 | 0.546 | 0.270 | 1.249 | 0.127 | 0.220 | 0.390 |
| 48 | butyrate | 94 | 7.249 | 6.728 | 3.245 | 0.335 | 4.872 | 9.504 |
| 0 | propionate | 96 | 0.362 | 0.162 | 1.047 | 0.107 | 0.117 | 0.230 |
| 48 | propionate | 94 | 5.076 | 4.750 | 2.191 | 0.226 | 3.436 | 6.679 |
| 0 | succinate | 96 | 0.280 | 0.140 | 0.914 | 0.093 | 0.000 | 0.246 |
| 48 | succinate | 94 | 1.741 | 1.402 | 1.888 | 0.195 | 0.000 | 3.020 |
## Combined summary table contains 60 condition combinations
## Combined summary saved to results/combined_summary_statistics.csv
## Subject-level and case-only analyses saved to results/ directory
We performed several statistical tests to compare SCFA concentrations across different experimental conditions. The following tables summarize the results of these comparisons, including t-tests for group differences and ANOVA for carbohydrate effects.
This table presents the results of t-tests comparing SCFA concentrations between the control and case groups.
| Analyte | .y. | group1 | group2 | n1 | n2 | statistic | df | P-value | Adjusted P-value | Significance |
|---|---|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | concentration | control | case | 96 | 94 | 0.8637 | 183.9200 | 0.3890 | 0.6338 | ns |
| acetate | concentration | control | case | 96 | 94 | 0.6641 | 187.9107 | 0.5070 | 0.6338 | ns |
| butyrate | concentration | control | case | 96 | 94 | 1.1509 | 185.8162 | 0.2510 | 0.6275 | ns |
| propionate | concentration | control | case | 96 | 94 | 0.2002 | 187.2715 | 0.8420 | 0.8420 | ns |
| succinate | concentration | control | case | 96 | 94 | 2.1520 | 170.4459 | 0.0328 | 0.1640 | ns |
This table shows the results of ANOVA tests examining the effect of different carbohydrate types on SCFA concentrations.
| Analyte | Effect | DFn | DFd | F | P-value | p<.05 | ges | Adjusted P-value | Significance |
|---|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | carbohydrate_type | 2 | 187 | 0.196 | 0.822 | 0.002 | 0.8220 | ns | |
| acetate | carbohydrate_type | 2 | 187 | 0.919 | 0.401 | 0.010 | 0.5012 | ns | |
| butyrate | carbohydrate_type | 2 | 187 | 1.791 | 0.170 | 0.019 | 0.4167 | ns | |
| propionate | carbohydrate_type | 2 | 187 | 1.398 | 0.250 | 0.015 | 0.4167 | ns | |
| succinate | carbohydrate_type | 2 | 187 | 2.555 | 0.080 | 0.027 | 0.4000 | ns |
Following the ANOVA, pairwise t-tests were performed to compare each carbohydrate type to the ‘no carbohydrate’ control. The results are shown below.
| Analyte | .y. | group1 | group2 | n1 | n2 | P-value | p.signif | Adjusted P-value | Significance |
|---|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | concentration | no_carbohydrate | rapid_digestible | 62 | 64 | 0.5390 | ns | 0.5989 | ns |
| 5aminovalerate | concentration | no_carbohydrate | slow_digestible | 62 | 64 | 0.6780 | ns | 0.6780 | ns |
| acetate | concentration | no_carbohydrate | rapid_digestible | 62 | 64 | 0.4020 | ns | 0.5743 | ns |
| acetate | concentration | no_carbohydrate | slow_digestible | 62 | 64 | 0.1810 | ns | 0.3620 | ns |
| butyrate | concentration | no_carbohydrate | rapid_digestible | 62 | 64 | 0.2390 | ns | 0.3983 | ns |
| butyrate | concentration | no_carbohydrate | slow_digestible | 62 | 64 | 0.0625 | ns | 0.3125 | ns |
| propionate | concentration | no_carbohydrate | rapid_digestible | 62 | 64 | 0.0991 | ns | 0.3303 | ns |
| propionate | concentration | no_carbohydrate | slow_digestible | 62 | 64 | 0.5150 | ns | 0.5989 | ns |
| succinate | concentration | no_carbohydrate | rapid_digestible | 62 | 64 | 0.1350 | ns | 0.3375 | ns |
| succinate | concentration | no_carbohydrate | slow_digestible | 62 | 64 | 0.0277 |
|
0.2770 | ns |
To assess the combined effects of group, carbohydrate type, and time, a three-way ANOVA was conducted. The results are summarized in this table.
| Analyte | Effect | P-value | Adjusted P-value | Significance |
|---|---|---|---|---|
| 5aminovalerate | group | 0.280 | 0.5158 | ns |
| 5aminovalerate | carbohydrate_type | 0.532 | 0.7758 | ns |
| 5aminovalerate | timepoint_hr | 0.000 | 0.0000 | **** |
| 5aminovalerate | group:carbohydrate_type | 0.905 | 0.9800 | ns |
| 5aminovalerate | group:timepoint_hr | 0.747 | 0.9338 | ns |
| 5aminovalerate | carbohydrate_type:timepoint_hr | 0.600 | 0.8400 | ns |
| 5aminovalerate | group:carbohydrate_type:timepoint_hr | 0.915 | 0.9800 | ns |
| acetate | group | 0.182 | 0.3981 | ns |
| acetate | carbohydrate_type | 0.020 | 0.0636 | ns |
| acetate | timepoint_hr | 0.000 | 0.0000 | **** |
| acetate | group:carbohydrate_type | 0.349 | 0.6107 | ns |
| acetate | group:timepoint_hr | 0.245 | 0.4764 | ns |
| acetate | carbohydrate_type:timepoint_hr | 0.079 | 0.1975 | ns |
| acetate | group:carbohydrate_type:timepoint_hr | 0.668 | 0.8659 | ns |
| butyrate | group | 0.058 | 0.1562 | ns |
| butyrate | carbohydrate_type | 0.010 | 0.0389 |
|
| butyrate | timepoint_hr | 0.000 | 0.0000 | **** |
| butyrate | group:carbohydrate_type | 0.511 | 0.7758 | ns |
| butyrate | group:timepoint_hr | 0.644 | 0.8659 | ns |
| butyrate | carbohydrate_type:timepoint_hr | 0.008 | 0.0350 |
|
| butyrate | group:carbohydrate_type:timepoint_hr | 0.489 | 0.7758 | ns |
| propionate | group | 0.949 | 0.9800 | ns |
| propionate | carbohydrate_type | 0.007 | 0.0350 |
|
| propionate | timepoint_hr | 0.000 | 0.0000 | **** |
| propionate | group:carbohydrate_type | 0.975 | 0.9800 | ns |
| propionate | group:timepoint_hr | 0.180 | 0.3981 | ns |
| propionate | carbohydrate_type:timepoint_hr | 0.008 | 0.0350 |
|
| propionate | group:carbohydrate_type:timepoint_hr | 0.957 | 0.9800 | ns |
| succinate | group | 0.018 | 0.0630 | ns |
| succinate | carbohydrate_type | 0.049 | 0.1429 | ns |
| succinate | timepoint_hr | 0.000 | 0.0000 | **** |
| succinate | group:carbohydrate_type | 0.980 | 0.9800 | ns |
| succinate | group:timepoint_hr | 0.440 | 0.7333 | ns |
| succinate | carbohydrate_type:timepoint_hr | 0.214 | 0.4406 | ns |
| succinate | group:carbohydrate_type:timepoint_hr | 0.879 | 0.9800 | ns |
To account for individual variability, we conducted analyses at the subject level. This allows us to examine within-subject changes and summarize statistics for each participant, providing a more granular view of the data.
This table provides summary statistics for each subject, including the mean concentration and number of observations.
| Group | Analyte | n Subjects | Mean Subject Means | SD Subject Means | SEM Subjects |
|---|---|---|---|---|---|
| control | 5aminovalerate | 16 | 0.549 | 0.156 | 0.039 |
| control | acetate | 16 | 18.722 | 6.602 | 1.651 |
| control | butyrate | 16 | 4.205 | 1.642 | 0.410 |
| control | propionate | 16 | 2.736 | 1.157 | 0.289 |
| control | succinate | 16 | 1.254 | 1.206 | 0.302 |
| case | 5aminovalerate | 16 | 0.470 | 0.305 | 0.076 |
| case | acetate | 16 | 16.983 | 3.150 | 0.787 |
| case | butyrate | 16 | 3.529 | 1.105 | 0.276 |
| case | propionate | 16 | 2.646 | 0.979 | 0.245 |
| case | succinate | 16 | 0.771 | 0.742 | 0.186 |
This table shows the results of statistical tests on the changes in SCFA concentrations within each subject from baseline to 48 hours.
| Group | Carbohydrate Type | Analyte | n | Mean Change | SD Change | SEM Change | t-statistic | P-value | Adjusted P-value | Significance |
|---|---|---|---|---|---|---|---|---|---|---|
| control | no_carbohydrate | 5aminovalerate | 16 | 0.9675 | 0.5707 | 0.1427 | 6.7813 | 0.0000 | 0.0000 | *** |
| control | no_carbohydrate | acetate | 16 | 26.0637 | 9.8419 | 2.4605 | 10.5930 | 0.0000 | 0.0000 | *** |
| control | no_carbohydrate | butyrate | 16 | 4.8569 | 2.2522 | 0.5631 | 8.6259 | 0.0000 | 0.0000 | *** |
| control | no_carbohydrate | propionate | 16 | 5.2625 | 2.3009 | 0.5752 | 9.1486 | 0.0000 | 0.0000 | *** |
| control | no_carbohydrate | succinate | 16 | 1.2794 | 1.2816 | 0.3204 | 3.9931 | 0.0012 | 0.0055 | ** |
| control | rapid_digestible | 5aminovalerate | 16 | 0.7809 | 0.3272 | 0.0818 | 9.5458 | 0.0000 | 0.0000 | *** |
| control | rapid_digestible | acetate | 16 | 31.2150 | 12.5947 | 3.1487 | 9.9137 | 0.0000 | 0.0000 | *** |
| control | rapid_digestible | butyrate | 16 | 7.2094 | 4.5932 | 1.1483 | 6.2783 | 0.0000 | 0.0000 | *** |
| control | rapid_digestible | propionate | 16 | 3.3881 | 2.5521 | 0.6380 | 5.3104 | 0.0001 | 0.0001 | *** |
| control | rapid_digestible | succinate | 16 | 1.5428 | 2.2543 | 0.5636 | 2.7376 | 0.0153 | 0.0183 |
|
| control | slow_digestible | 5aminovalerate | 16 | 0.8838 | 0.4453 | 0.1113 | 7.9380 | 0.0000 | 0.0000 | *** |
| control | slow_digestible | acetate | 16 | 32.3325 | 11.6928 | 2.9232 | 11.0607 | 0.0000 | 0.0000 | *** |
| control | slow_digestible | butyrate | 16 | 8.4188 | 3.9175 | 0.9794 | 8.5960 | 0.0000 | 0.0000 | *** |
| control | slow_digestible | propionate | 16 | 4.5875 | 2.6021 | 0.6505 | 7.0520 | 0.0000 | 0.0000 | *** |
| control | slow_digestible | succinate | 16 | 1.9950 | 2.3006 | 0.5752 | 3.4686 | 0.0034 | 0.0055 | ** |
| case | no_carbohydrate | 5aminovalerate | 14 | 0.9021 | 0.4506 | 0.1204 | 7.4913 | 0.0000 | 0.0000 | *** |
| case | no_carbohydrate | acetate | 14 | 30.2914 | 4.7348 | 1.2654 | 23.9375 | 0.0000 | 0.0000 | *** |
| case | no_carbohydrate | butyrate | 14 | 5.7114 | 1.3488 | 0.3605 | 15.8437 | 0.0000 | 0.0000 | *** |
| case | no_carbohydrate | propionate | 14 | 6.0179 | 1.8770 | 0.5017 | 11.9961 | 0.0000 | 0.0000 | *** |
| case | no_carbohydrate | succinate | 14 | 0.6471 | 1.2413 | 0.3317 | 1.9507 | 0.0730 | 0.0730 | ns |
| case | rapid_digestible | 5aminovalerate | 16 | 0.8112 | 0.6987 | 0.1747 | 4.6443 | 0.0003 | 0.0003 | *** |
| case | rapid_digestible | acetate | 16 | 31.0581 | 7.0327 | 1.7582 | 17.6650 | 0.0000 | 0.0000 | *** |
| case | rapid_digestible | butyrate | 16 | 6.5300 | 3.9291 | 0.9823 | 6.6478 | 0.0000 | 0.0000 | *** |
| case | rapid_digestible | propionate | 16 | 4.1556 | 1.6021 | 0.4005 | 10.3756 | 0.0000 | 0.0000 | *** |
| case | rapid_digestible | succinate | 16 | 1.4047 | 1.6331 | 0.4083 | 3.4405 | 0.0036 | 0.0055 | ** |
| case | slow_digestible | 5aminovalerate | 16 | 0.8075 | 0.6879 | 0.1720 | 4.6951 | 0.0003 | 0.0003 | *** |
| case | slow_digestible | acetate | 16 | 35.7078 | 6.5142 | 1.6286 | 21.9261 | 0.0000 | 0.0000 | *** |
| case | slow_digestible | butyrate | 16 | 7.3503 | 2.5373 | 0.6343 | 11.5875 | 0.0000 | 0.0000 | *** |
| case | slow_digestible | propionate | 16 | 5.0372 | 2.7809 | 0.6952 | 7.2455 | 0.0000 | 0.0000 | *** |
| case | slow_digestible | succinate | 16 | 1.7725 | 1.8763 | 0.4691 | 3.7788 | 0.0018 | 0.0055 | ** |
To isolate the effects of the intervention within the case group, we performed a temporal analysis comparing SCFA concentrations at 0h and 48h. This analysis helps to understand the direct impact of the carbohydrate types on the case subjects over time.
This table presents the temporal changes in SCFA concentrations for the case group, broken down by carbohydrate type.
| Carbohydrate Type | Analyte | n | Mean Change | SD Change | SEM Change | t-statistic | P-value | Adjusted P-value | Significance |
|---|---|---|---|---|---|---|---|---|---|
| no_carbohydrate | 5aminovalerate | 14 | 0.9021 | 0.4506 | 0.1204 | 7.4913 | 0.0000 | 0.0000 | *** |
| no_carbohydrate | acetate | 14 | 30.2914 | 4.7348 | 1.2654 | 23.9375 | 0.0000 | 0.0000 | *** |
| no_carbohydrate | butyrate | 14 | 5.7114 | 1.3488 | 0.3605 | 15.8437 | 0.0000 | 0.0000 | *** |
| no_carbohydrate | propionate | 14 | 6.0179 | 1.8770 | 0.5017 | 11.9961 | 0.0000 | 0.0000 | *** |
| no_carbohydrate | succinate | 14 | 0.6471 | 1.2413 | 0.3317 | 1.9507 | 0.0730 | 0.0730 | ns |
| rapid_digestible | 5aminovalerate | 16 | 0.8112 | 0.6987 | 0.1747 | 4.6443 | 0.0003 | 0.0003 | *** |
| rapid_digestible | acetate | 16 | 31.0581 | 7.0327 | 1.7582 | 17.6650 | 0.0000 | 0.0000 | *** |
| rapid_digestible | butyrate | 16 | 6.5300 | 3.9291 | 0.9823 | 6.6478 | 0.0000 | 0.0000 | *** |
| rapid_digestible | propionate | 16 | 4.1556 | 1.6021 | 0.4005 | 10.3756 | 0.0000 | 0.0000 | *** |
| rapid_digestible | succinate | 16 | 1.4047 | 1.6331 | 0.4083 | 3.4405 | 0.0036 | 0.0055 | ** |
| slow_digestible | 5aminovalerate | 16 | 0.8075 | 0.6879 | 0.1720 | 4.6951 | 0.0003 | 0.0003 | *** |
| slow_digestible | acetate | 16 | 35.7078 | 6.5142 | 1.6286 | 21.9261 | 0.0000 | 0.0000 | *** |
| slow_digestible | butyrate | 16 | 7.3503 | 2.5373 | 0.6343 | 11.5875 | 0.0000 | 0.0000 | *** |
| slow_digestible | propionate | 16 | 5.0372 | 2.7809 | 0.6952 | 7.2455 | 0.0000 | 0.0000 | *** |
| slow_digestible | succinate | 16 | 1.7725 | 1.8763 | 0.4691 | 3.7788 | 0.0018 | 0.0055 | ** |
Here, we present the temporal changes for the case group, pooled across all carbohydrate types to assess the overall time effect.
| Analyte | n | Mean Change | SD Change | SEM Change | t-statistic | P-value | Adjusted P-value | Significance |
|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | 46 | 0.8376 | 0.6173 | 0.0910 | 9.2035 | 0 | 0 | *** |
| acetate | 46 | 32.4421 | 6.5589 | 0.9671 | 33.5470 | 0 | 0 | *** |
| butyrate | 46 | 6.5662 | 2.8747 | 0.4239 | 15.4917 | 0 | 0 | *** |
| propionate | 46 | 5.0290 | 2.2420 | 0.3306 | 15.2134 | 0 | 0 | *** |
| succinate | 46 | 1.3021 | 1.6503 | 0.2433 | 5.3512 | 0 | 0 | *** |
This table summarizes the results from the mixed-effects models applied to the case group data, accounting for subject-specific random effects.
| Analyte | Effect | F-value | P-value | Significance |
|---|---|---|---|---|
| acetate | timepoint_hr | 2000.3155 | 0.0000 | *** |
| acetate | carbohydrate_type | 5.5775 | 0.0054 | ** |
| acetate | timepoint_hr:carbohydrate_type | 5.6507 | 0.0051 | ** |
| butyrate | timepoint_hr | 322.1146 | 0.0000 | *** |
| butyrate | carbohydrate_type | 1.5130 | 0.2266 | ns |
| butyrate | timepoint_hr:carbohydrate_type | 1.5034 | 0.2287 | ns |
| propionate | timepoint_hr | 382.4372 | 0.0000 | *** |
| propionate | carbohydrate_type | 4.8293 | 0.0105 |
|
| propionate | timepoint_hr:carbohydrate_type | 4.3048 | 0.0168 |
|
| 5aminovalerate | timepoint_hr | 118.6212 | 0.0000 | *** |
| 5aminovalerate | carbohydrate_type | 0.1080 | 0.8978 | ns |
| 5aminovalerate | timepoint_hr:carbohydrate_type | 0.0982 | 0.9066 | ns |
| succinate | timepoint_hr | 47.8222 | 0.0000 | *** |
| succinate | carbohydrate_type | 2.6806 | 0.0749 | ns |
| succinate | timepoint_hr:carbohydrate_type | 2.3631 | 0.1009 | ns |
To further investigate the magnitude of change over time, we calculated the delta (change) in concentration for each analyte between 48h and 0h. This approach focuses on the response magnitude and allows us to test whether the group or carbohydrate type influences how strongly subjects respond to the intervention over time.
This table provides summary statistics for the calculated delta values (48h - 0h), showing the mean change and variability.
| Group | Carbohydrate Type | Analyte | n | Mean Delta | SD Delta | SEM Delta |
|---|---|---|---|---|---|---|
| control | no_carbohydrate | 5aminovalerate | 16 | 0.967 | 0.571 | 0.143 |
| control | no_carbohydrate | acetate | 16 | 26.064 | 9.842 | 2.460 |
| control | no_carbohydrate | butyrate | 16 | 4.857 | 2.252 | 0.563 |
| control | no_carbohydrate | propionate | 16 | 5.263 | 2.301 | 0.575 |
| control | no_carbohydrate | succinate | 16 | 1.279 | 1.282 | 0.320 |
| control | rapid_digestible | 5aminovalerate | 16 | 0.781 | 0.327 | 0.082 |
| control | rapid_digestible | acetate | 16 | 31.215 | 12.595 | 3.149 |
| control | rapid_digestible | butyrate | 16 | 7.209 | 4.593 | 1.148 |
| control | rapid_digestible | propionate | 16 | 3.388 | 2.552 | 0.638 |
| control | rapid_digestible | succinate | 16 | 1.543 | 2.254 | 0.564 |
| control | slow_digestible | 5aminovalerate | 16 | 0.884 | 0.445 | 0.111 |
| control | slow_digestible | acetate | 16 | 32.333 | 11.693 | 2.923 |
| control | slow_digestible | butyrate | 16 | 8.419 | 3.918 | 0.979 |
| control | slow_digestible | propionate | 16 | 4.588 | 2.602 | 0.651 |
| control | slow_digestible | succinate | 16 | 1.995 | 2.301 | 0.575 |
| case | no_carbohydrate | 5aminovalerate | 14 | 0.902 | 0.451 | 0.120 |
| case | no_carbohydrate | acetate | 14 | 30.291 | 4.735 | 1.265 |
| case | no_carbohydrate | butyrate | 14 | 5.711 | 1.349 | 0.360 |
| case | no_carbohydrate | propionate | 14 | 6.018 | 1.877 | 0.502 |
| case | no_carbohydrate | succinate | 14 | 0.647 | 1.241 | 0.332 |
| case | rapid_digestible | 5aminovalerate | 16 | 0.811 | 0.699 | 0.175 |
| case | rapid_digestible | acetate | 16 | 31.058 | 7.033 | 1.758 |
| case | rapid_digestible | butyrate | 16 | 6.530 | 3.929 | 0.982 |
| case | rapid_digestible | propionate | 16 | 4.156 | 1.602 | 0.401 |
| case | rapid_digestible | succinate | 16 | 1.405 | 1.633 | 0.408 |
| case | slow_digestible | 5aminovalerate | 16 | 0.807 | 0.688 | 0.172 |
| case | slow_digestible | acetate | 16 | 35.708 | 6.514 | 1.629 |
| case | slow_digestible | butyrate | 16 | 7.350 | 2.537 | 0.634 |
| case | slow_digestible | propionate | 16 | 5.037 | 2.781 | 0.695 |
| case | slow_digestible | succinate | 16 | 1.772 | 1.876 | 0.469 |
This table shows the results of t-tests comparing the delta values between the control and case groups.
| analyte | .y. | group1 | group2 | n1 | n2 | statistic | df | p | p.adj | p.adj.signif |
|---|---|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | delta_48h_0h | control | case | 48 | 46 | 0.3544 | 82.6540 | 0.724 | 0.7240 | ns |
| acetate | delta_48h_0h | control | case | 48 | 46 | -1.3367 | 75.1760 | 0.185 | 0.5425 | ns |
| butyrate | delta_48h_0h | control | case | 48 | 46 | 0.3699 | 86.0520 | 0.712 | 0.7240 | ns |
| propionate | delta_48h_0h | control | case | 48 | 46 | -1.2437 | 91.2871 | 0.217 | 0.5425 | ns |
| succinate | delta_48h_0h | control | case | 48 | 46 | 0.8088 | 90.2737 | 0.421 | 0.7017 | ns |
This table presents the results of ANOVA tests on the delta values to examine the effect of carbohydrate type on the magnitude of change.
| analyte | Effect | DFn | DFd | F | p | p<.05 | ges | p.adj | p.adj.signif |
|---|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | carbohydrate_type | 2 | 91 | 0.538 | 0.586 | 0.012 | 0.5860 | ns | |
| acetate | carbohydrate_type | 2 | 91 | 3.243 | 0.044 |
|
0.067 | 0.0733 | ns |
| butyrate | carbohydrate_type | 2 | 91 | 4.968 | 0.009 |
|
0.098 | 0.0225 |
|
| propionate | carbohydrate_type | 2 | 91 | 4.932 | 0.009 |
|
0.098 | 0.0225 |
|
| succinate | carbohydrate_type | 2 | 91 | 1.923 | 0.152 | 0.041 | 0.1900 | ns |
Following the ANOVA on delta values, pairwise t-tests were performed to compare each carbohydrate type to the ‘no carbohydrate’ control. The results are shown below.
| analyte | .y. | group1 | group2 | n1 | n2 | p | p.signif | p.adj | p.adj.signif |
|---|---|---|---|---|---|---|---|---|---|
| 5aminovalerate | delta_48h_0h | no_carbohydrate | rapid_digestible | 30 | 32 | 0.3080 | ns | 0.3422 | ns |
| 5aminovalerate | delta_48h_0h | no_carbohydrate | slow_digestible | 30 | 32 | 0.5080 | ns | 0.5080 | ns |
| acetate | delta_48h_0h | no_carbohydrate | rapid_digestible | 30 | 32 | 0.1900 | ns | 0.2714 | ns |
| acetate | delta_48h_0h | no_carbohydrate | slow_digestible | 30 | 32 | 0.0126 |
|
0.0420 |
|
| butyrate | delta_48h_0h | no_carbohydrate | rapid_digestible | 30 | 32 | 0.0576 | ns | 0.1152 | ns |
| butyrate | delta_48h_0h | no_carbohydrate | slow_digestible | 30 | 32 | 0.0023 | ** | 0.0119 |
|
| propionate | delta_48h_0h | no_carbohydrate | rapid_digestible | 30 | 32 | 0.0024 | ** | 0.0119 |
|
| propionate | delta_48h_0h | no_carbohydrate | slow_digestible | 30 | 32 | 0.1770 | ns | 0.2714 | ns |
| succinate | delta_48h_0h | no_carbohydrate | rapid_digestible | 30 | 32 | 0.2890 | ns | 0.3422 | ns |
| succinate | delta_48h_0h | no_carbohydrate | slow_digestible | 30 | 32 | 0.0530 | ns | 0.1152 | ns |
To assess the combined effects of group and carbohydrate type on the delta values, a two-way ANOVA was conducted. The results are summarized in this table.
| Analyte | Effect | P-value | Adjusted P-value | Significance |
|---|---|---|---|---|
| 5aminovalerate | group | 0.748 | 0.9350 | ns |
| 5aminovalerate | carbohydrate_type | 0.601 | 0.8700 | ns |
| 5aminovalerate | group:carbohydrate_type | 0.914 | 0.9540 | ns |
| acetate | group | 0.205 | 0.5125 | ns |
| acetate | carbohydrate_type | 0.048 | 0.2400 | ns |
| acetate | group:carbohydrate_type | 0.612 | 0.8700 | ns |
| butyrate | group | 0.638 | 0.8700 | ns |
| butyrate | carbohydrate_type | 0.009 | 0.0675 | ns |
| butyrate | group:carbohydrate_type | 0.492 | 0.8700 | ns |
| propionate | group | 0.177 | 0.5125 | ns |
| propionate | carbohydrate_type | 0.009 | 0.0675 | ns |
| propionate | group:carbohydrate_type | 0.954 | 0.9540 | ns |
| succinate | group | 0.392 | 0.8400 | ns |
| succinate | carbohydrate_type | 0.152 | 0.5125 | ns |
| succinate | group:carbohydrate_type | 0.852 | 0.9540 | ns |
This table summarizes the results from the mixed-effects models applied to the delta values, accounting for subject-specific random effects.
| Analyte | Effect | F-value | P-value | Significance |
|---|---|---|---|---|
| acetate | group | 0.6499 | 0.4261 | ns |
| acetate | carbohydrate_type | 19.3531 | 0.0000 | *** |
| acetate | group:carbohydrate_type | 2.7297 | 0.0731 | ns |
| butyrate | group | 0.0397 | 0.8433 | ns |
| butyrate | carbohydrate_type | 11.0901 | 0.0001 | *** |
| butyrate | group:carbohydrate_type | 2.5781 | 0.0840 | ns |
| propionate | group | 0.8998 | 0.3499 | ns |
| propionate | carbohydrate_type | 20.2457 | 0.0000 | *** |
| propionate | group:carbohydrate_type | 0.2458 | 0.7828 | ns |
| 5aminovalerate | group | 0.1182 | 0.7332 | ns |
| 5aminovalerate | carbohydrate_type | 0.7432 | 0.4798 | ns |
| 5aminovalerate | group:carbohydrate_type | 0.3313 | 0.7192 | ns |
| succinate | group | 0.1141 | 0.7377 | ns |
| succinate | carbohydrate_type | 6.2006 | 0.0035 | ** |
| succinate | group:carbohydrate_type | 0.0367 | 0.9639 | ns |
To account for the repeated measures design and the variability between subjects, we utilized linear mixed-effects models. These models include subject as a random effect, allowing us to more accurately assess the fixed effects of group, carbohydrate type, and time.
This table presents a summary of the full mixed-effects models, including F-values and p-values for all fixed effects.
| Analyte | Effect | F-value | P-value | Significance |
|---|---|---|---|---|
| acetate | group | 0.6249 | 0.4350 | ns |
| acetate | carbohydrate_type | 7.0955 | 0.0011 | ** |
| acetate | timepoint_hr | 1531.5721 | 0.0000 | *** |
| acetate | group:carbohydrate_type | 1.8474 | 0.1610 | ns |
| acetate | group:timepoint_hr | 2.3717 | 0.1255 | ns |
| acetate | carbohydrate_type:timepoint_hr | 4.5563 | 0.0119 |
|
| acetate | group:carbohydrate_type:timepoint_hr | 0.6918 | 0.5022 | ns |
| butyrate | group | 1.6308 | 0.2108 | ns |
| butyrate | carbohydrate_type | 6.4119 | 0.0021 | ** |
| butyrate | timepoint_hr | 537.8258 | 0.0000 | *** |
| butyrate | group:carbohydrate_type | 1.1091 | 0.3324 | ns |
| butyrate | group:timepoint_hr | 0.2140 | 0.6443 | ns |
| butyrate | carbohydrate_type:timepoint_hr | 6.5577 | 0.0018 | ** |
| butyrate | group:carbohydrate_type:timepoint_hr | 1.1286 | 0.3261 | ns |
| propionate | group | 0.0001 | 0.9907 | ns |
| propionate | carbohydrate_type | 8.1529 | 0.0004 | *** |
| propionate | timepoint_hr | 591.8170 | 0.0000 | *** |
| propionate | group:carbohydrate_type | 0.0493 | 0.9519 | ns |
| propionate | group:timepoint_hr | 2.8878 | 0.0912 | ns |
| propionate | carbohydrate_type:timepoint_hr | 7.7057 | 0.0006 | *** |
| propionate | group:carbohydrate_type:timepoint_hr | 0.0751 | 0.9276 | ns |
| 5aminovalerate | group | 0.6495 | 0.4262 | ns |
| 5aminovalerate | carbohydrate_type | 0.7869 | 0.4570 | ns |
| 5aminovalerate | timepoint_hr | 316.5623 | 0.0000 | *** |
| 5aminovalerate | group:carbohydrate_type | 0.1765 | 0.8384 | ns |
| 5aminovalerate | group:timepoint_hr | 0.1997 | 0.6556 | ns |
| 5aminovalerate | carbohydrate_type:timepoint_hr | 0.6132 | 0.5429 | ns |
| 5aminovalerate | group:carbohydrate_type:timepoint_hr | 0.1464 | 0.8640 | ns |
| succinate | group | 2.0132 | 0.1656 | ns |
| succinate | carbohydrate_type | 4.7476 | 0.0099 | ** |
| succinate | timepoint_hr | 79.7731 | 0.0000 | *** |
| succinate | group:carbohydrate_type | 0.0891 | 0.9148 | ns |
| succinate | group:timepoint_hr | 0.7792 | 0.3787 | ns |
| succinate | carbohydrate_type:timepoint_hr | 2.2732 | 0.1063 | ns |
| succinate | group:carbohydrate_type:timepoint_hr | 0.1141 | 0.8922 | ns |
To visually explore the data, we generated a series of plots. These visualizations illustrate the relationships between SCFA concentrations and the experimental variables, including group, carbohydrate type, and time. Each plot is designed to highlight different aspects of the data, from overall trends to individual subject responses.